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Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra)
J. Comp. Path. 2016, Vol. -, 1e6
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra) C. Salvadori*, J. Finlayson†, T. Trogu‡, N. Formenti‡, P. Lanfranchi‡, C. Citteriox, J. Palarea-Albaladejok, A. Poli* and F. Chianini† * Department of Veterinary Sciences, Pisa University, Viale delle Piagge, Pisa, Italy, † Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Near Edinburgh, UK, ‡ Department of Veterinary Sciences and Public Health, Milan University, Via Festa del Perdono, Milano, x Istituto Zooprofilattico Sperimentale delle Venezie, Sezione di Belluno, Via M. Cappellari, Belluno, Italy and k Biomathematics and Statistics Scotland, JCMB, The King’s Buildings, Peter Guthrie Tait Road, Edinburgh, UK
Summary Immune system cell subsets in lymph nodes and spleen from alpine chamois (Rupicapra rupicapra subspecies rupicapra) living in the Italian Alps were characterized immunohistochemically. Seven primary antibodies (against human CD3, CD79acy, CD68, or ovine CD4, CD8, CD21 and gd T-cell receptor [TCR] epitopes) were tested on tissues fixed either in formalin or in zinc salts (ZS) and cross-reactivity with chamois immune cell epitopes was shown. ZS fixation allowed wider identification of immune cells, without the need for antigen retrieval. CD4+ and CD21+ cells were labelled only in ZS-fixed tissues. Reagents specific for human CD3, CD79 and CD68 antigens successfully detected chamois immune cells, both in ZS-fixed and formalin-fixed tissues. The reactivity and distribution of immune cells in lymph nodes and spleen were similar to those described in other domestic and wild ruminants. Results from this study may allow future investigation of the immune response and pathogenesis of diseases in the chamois. Ó 2016 Elsevier Ltd. All rights reserved. Keywords: alpine chamois; immune system cells; immunohistochemistry; zinc salts fixation
Alpine chamois (Rupicapra rupicapra subspecies rupicapra, Linnaeus 1758) is a wild ruminant living in the eastern Alps. Infectious diseases in chamois have raised the interest of scientists because they may represent a threat for the conservation of wildlife species and biodiversity (Daszak et al., 2000; Pioz et al., 2008). Moreover, chamois could represent a useful animal model to evaluate the complex balance between host and pathogens (especially parasites, but also viruses and bacteria). Population dynamics of chamois are influenced by pathogen-derived epidemics and investigations of the polymorphism of maCorrespondence to: F. Chianini (e-mail: Moredun.ac.uk). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2016.06.012
Francesca.Chianini@
jor histocompatibility complex (MHC) class II genes were used to study the adaptive processes and pathogen-mediated selection (Mona et al., 2008; Cavallero et al., 2012). However, evaluation of inflammatory infiltrates in tissues may be crucial to understanding of the pathogenesis of infectious diseases in this species. An immunohistochemical study of formalin-fixed tissue samples from healthy and mange-infected chamois failed to demonstrate reactivity of immune cells with a panel of antihuman antibodies (Rode et al., 1998) and no specific antibodies are available for detection of chamois immune cells. However, immunohistochemistry (IHC) is dependent on adequate fixation of the tissue. Routinely, tissues are fixed in 10% neutral buffered Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
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C. Salvadori et al.
formalin, which inhibits cellular processes and prevents tissue degradation, but is a limiting factor for IHC as it cross-links antigens and masks epitopes (Webster et al., 2009). Zinc salts (ZS) fixative is a non-aldehyde solution, composed of zinc acetate and zinc chloride in Tris-calcium acetate buffer, that is considered to preserve sensitive antigens similar to in frozen tissue, but with a morphological preservation comparable to formalin fixation (Beckstead, 1994). ZS fixation has been used successfully in immunohistochemical studies of ovine and cervine immune cells (Gonzalez et al., 2001; Dagleish et al., 2012). The aims of this study were: (1) to determine the cross-reactivity of anti-human and anti-ovine monoclonal antibodies with chamois immune cells in lymph nodes and spleen, and (2) to evaluate differences and effectiveness of immune cell labelling in chamois tissues fixed either in ZS or in formalin. Retromandibular lymph nodes and spleen were collected from 10 alpine chamois (seven females and three males), aged between 1 and 14 years, which were shot during the hunting season (from September to December 2013). A thin slice (4e5 mm thick) of each tissue sampled was immediately placed in freshly prepared ZS fixative for 24e48 h (Beckstead, 1994), and a duplicate sample was placed into 10% neutral buffered formalin for 48e72 h. All samples were processed routinely and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE). Serial sections were mounted on treated glass slides (Superfrost Plus; Menzel-Glaser, Braunschweig, Germany) for IHC. Different antigen retrieval methods were used for formalin-fixed tissues as shown in Supplementary Table 1. A panel of monoclonal antibodies (Supplementary Table 1) was applied and incubated overnight at 4 C. Antibody binding was detected by the EnVision PlusÔ System-HRP (DAB) (Dako UK Ltd., Ely, UK). Omission of the primary antibody was used to provide a negative control. Ovine lymph node sections were used as positive controls. Bright field images were acquired at 200 magnification with a Leica Microsystem DFC490 digital camera mounted on a Leica DMR microscope (Leica, Wetzlar, Germany). For each animal, five non-contiguous 10,000 mm2 fields of view were selected randomly. Labelled cells were counted in the following areas of those fields: the germinal centre and mantle zone of the follicles and the interfollicular/paracortical area and medullary cords of the lymph nodes, and the germinal centre and mantle zone of the follicles and the periarteriolar lymphoid sheaths, marginal zone and red pulp of the spleen. The number of total cells (total nuclei
evident with haematoxylin counterstaining) and CD3, CD79, CD68, CD21 and CD4-positive cells within a field were counted using a semi-automatic analysis system (LASV 4.3, Leica). The mean percentage of positively labelled cells and a 95% confidence interval computed by the AgrestieCoull method were obtained for each marker by area and fixation method. A generalized linear mixed model (GLMM) was fitted to test for significant differences in mean percentage of positive cells detected between the two fixation methods (i.e. formalin and ZS) for each organ, area and marker separately. Only markers detectable by both techniques were modelled. The GLMMs were fitted by maximum likelihood to the number of positive cells out of the total number of cells, with logit link function and binomially distributed errors, and using Laplace approximations to calculate log-likelihoods. Fixation method was included as a fixed effect and animal identification as a random effect in order to account for both within and between animal variability. The significance of the fixation method effect was assessed using P values derived from the likelihood ratio test at the usual 5% significance level. The data analyses and graphics were produced using the R system for statistical computing v3.2 (R Core Team, 2015). Good tissue morphology was evident in lymph nodes and spleen using either formalin or ZS fixation. Antibodies raised against human CD3, CD79 and CD68 epitopes reacted successfully with Alpine chamois immune cells, both in formalin- and ZSfixed tissues. Antibodies raised against ovine CD4 and CD21 labelled Alpine chamois cells only in ZSfixed tissues. Clone 7.2.38 of anti-CD3 antibody labelled the surface and cytoplasm of cells in the interfollicular/paracortical areas of lymph nodes (Supplementary Fig. 1A) and the periarteriolar lymphoid sheaths of the spleen (Supplementary Fig. 2A). A prevalence of CD3+ cells was evident in the marginal zone of the spleen and medullary cords of lymph nodes (Figs. 3 and 4). A moderate number of CD3+ cells was present in the mantle zone both in the lymph node and spleen, and few CD3+ cells were seen in the germinal centres of the follicles. No significant mean differences were detected in CD3 immunolabelling between formalin- and ZS-fixed lymph node (Fig. 1). In the spleen, significant differences were found only in the marginal zone, where ZS-fixed tissues showed a higher average percentage of labelled cells when compared with formalin-fixed tissues (64.65% versus 41.94%; P ¼ 0.024) (Fig. 2). Specific immunolabelling with anti-CD4 monoclonal antibody was observed only in ZS-fixed tissues (Supplementary Figs. 1B and 2B). CD4+ cells were
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
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Immune Cells in Fixed Tissue from Alpine Chamois Lymph nodes
CD3
CD79
CD68
100
ZSF Formalin
Mean % positive cells
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Fig. 1. Lymph nodes: mean percentages (%) of CD3 , CD79 and CD68+ cells detected by area and fixative.
lymphoid sheath of the spleen (60.97%) (Figs. 3 and 4). CD79+ cells were mainly localized in germinal centres and mantle zones of follicles in lymph nodes and spleen (Supplementary Figs. 1C and 2C).
small round cells that mirrored the distribution of CD3+ cells, although with less intense labelling. The highest mean percentages of CD4+ lymphocytes were in the interfollicular/paracortical areas of the lymph node (69.66%) and in the periarteriolar
Spleen
CD3
CD79
CD68 ZSF Formalin
Mean % positive cells
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Fig. 2. Spleen: mean percentages (%) of CD3 , CD79 and CD68 cells detected by area and fixative. PALS, periarteriolar lymphoid sheath.
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
C. Salvadori et al.
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Lymph nodes
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CD3 CD4 CD79 CD21 CD68
Mean % positive cells
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0 Germinal centre
Mantle zone
Paracortex
Medullary cords
Area
Fig. 3. Lymph nodes: mean percentages (%) of CD3+, CD4+, CD79+, CD21+ and CD68+ cells by area detected in ZS-fixed tissues.
Multifocally, CD79+ lymphocytes were also present in the medulla of lymph nodes and in the marginal zone of the spleen. In the medulla, labelled cells were mainly localized around small blood vessels, and multifocally, large cells with cytoplasmic label-
ling were evident. Smooth muscle cells and endothelial cells of small blood vessels occasionally reacted positively with anti-CD79 monoclonal antibody. Results of immunolabelling were variable between different areas of lymph node and spleen depending Spleen
100
CD3 CD4 CD79 CD21 CD68
Mean % positive cells
80
60
40
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0 Germinal centre
Mantle zone
Marginal zone
PALS
Red pulp
Area
Fig. 4. Spleen: mean percentages (%) of CD3+, CD4+, CD79+, CD21+ and CD68+ cells by area detected in ZS-fixed tissues. PALS, periarteriolar lymphoid sheath.
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
Immune Cells in Fixed Tissue from Alpine Chamois
on the fixative (Figs. 1 and 2). The mean differences were significant, with formalin fixation detecting, on average, a significantly higher percentage of CD79+ cells in the germinal centres of lymph nodes (87.47% versus 68.25%; P <0.001) and spleen (91.38% versus 79.56%; P <0.001), and in the medullary cords of lymph nodes (10.92% versus 4.42%; P ¼ 0.016), and the periarteriolar lymphoid sheath (3.90% versus 1.17%; P <0.001) and the red pulp (4.36% versus 1.82%; P ¼ 0.005) of the spleen. Conversely, ZS fixation revealed the highest percentages of CD79+ cells in the interfollicular/paracortical area (4.25% versus 2.27%; P ¼ 0.003) and mantle zone (46.46% versus 38.64%; P ¼ 0.011) of the lymph node. No significant differences in the mean number of labelled cells were found for the mantle and marginal zones of the spleen. Anti-CD21 antibody labelled small round cells and, mainly, cells with a reticular pattern, in the light zone of the germinal centre and mantle zones of the lymph nodes and the splenic follicles (from 99.66% in the mantle zone of the spleen to 97.39% in the mantle zone of the lymph node) (Supplementary Figs. 1D and 2D). The distribution of CD21+ cells was similar to that of CD79+ cells; however, in all considered areas, the percentages of CD21+ cells were higher, on average, than those of CD79+ cells (Figs. 3 and 4). An intracytoplasmic granular pattern of labelling was observed for cells consistent with macrophages using monoclonal anti-human CD68 antibody (Supplementary Figs. 1F and 2F). CD68+ cells were localized mainly in the medullary cords of the lymph nodes and the marginal zone and red pulp of the spleen (Supplementary Figs. 1E and 2E). ZS fixation provided the highest average percentage of CD68+ cells in the germinal centres (12.53% versus 10.77%; P ¼ 0.040) and interfollicular/paracortical areas (7.62% versus 1.40%; P <0.001) of the lymph nodes and in the germinal centres (11.11% versus 4.38%; P <0.001) and the marginal zones (44.59% versus 35.43%; P ¼ 0.042) of the spleen. Conversely, formalin fixation revealed significantly higher mean percentages of CD68+ cells in the medullary cords (32.29% versus 20.41%; P ¼ 0.029) of the lymph nodes and the red pulp (62.45% versus 49.46%; P ¼ 0.028) of the spleen. In the other areas (i.e. the mantle zone of the lymph node and the mantle zone and periarteriolar lymphoid sheath of the spleen), there were no significant differences in the mean percentages of labelled cells between fixatives (Figs. 1 and 2). No specific labelling was detected using several clones against CD8 and gd T-cell receptor (TCR) antigens in ZS- or formalin-fixed tissues, with or without antigen retrieval. Control ovine lymph node showed
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that all antibodies labelled the expected cell populations, including CD8+ T cells and gd TCR+ cells. No labelling was detected in the negative control slides. This study provides the first description of immune system cell subsets in Alpine chamois lymph nodes and spleen. Histology of the lymph node and spleen was similar to that of other animals (Galeotti et al., 1993; Navarro et al., 1996; Gonzalez et al., 2001; Cesta, 2006; Drayton et al., 2006; Willard-Mack, 2006; Dagleish et al., 2012). The reactivity and distribution of immune cells in lymph nodes and spleen were similar to those described in goats (Navarro et al., 1996), sheep (Gonzalez et al., 2001), cattle (Galeotti et al., 1993) and deer (Dagleish et al., 2012). Monoclonal antibodies specific for human CD3, CD79 and CD68 appeared to cross-react with Alpine chamois antigens in the lymph nodes and spleen, with tissues prepared using both fixatives. Antigen-retrieval methods, which can be difficult to standardize and are time consuming, were necessary for formalin-fixed tissues in order to reverse the detrimental effects of fixation. As reported in previous immunohistochemical studies carried out on ovine and cervine tissues (Gonzalez et al., 2001; Dagleish et al., 2012), ZS fixation allowed good preservation of tissue morphology and a wider detection of cell surface epitopes compared with formalin fixation. These results could be related to the efficiency of ZS fixative, which does not cause conformational changes in the tertiary and quaternary structures of proteins, leaving the surface receptors available for the interaction with antibody. Secondary changes in the tissues after antigen retrieval treatment, which can cause rarefaction and detachment of the cells from the slides, should be taken into account in assessing the differences of immunolabelling between formalin and ZS fixation. The absence of reaction with monoclonal antibodies specific for CD8 and gd TCR was similar to results seen in cervine tissues (Dagleish et al., 2012). These antigens seem to be extremely sensitive to fixation and heating (Tingstedt et al., 2003; Dagleish et al., 2012). In conclusion, ZS appears to be an effective fixative that allows good preservation of surface receptors and specific immunolabelling for Alpine chamois lymphoid tissues. This provides a basis for future studies on the pathogenesis of diseases and immune response in this species.
Acknowledgments The authors thank M. Vascellari, Istituto Zooprofilattico Sperimentale delle Venezie, for tissue processing of ZS-fixed specimens. This work was funded by
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
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PRIN (Programmi di Ricerca di Interesse Nazionale) 2010P7LFW4_004 from the Italian Ministero dell’Istruzione, dell’Universita e della Ricerca (MIUR) and by the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS).
Conflict of Interest Statement All authors have no conflicts of interest to declare related to the publication of this manuscript.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jcpa.2016.06.012.
References Beckstead JH (1994) A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues. Journal of Histochemistry and Cytochemistry, 42, 1127e1134. Cavallero S, Marco I, Lavın S, D’Amelio S, L opezOlvera JR (2012) Polymorphisms at MHC class II DRB1 exon 2 locus in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Infection Genetics and Evolution, 12, 1020e1026. Cesta MF (2006) Normal structure, function, and histology of the spleen. Toxicologic Pathology, 34, 455e465. Dagleish MP, Finlayson J, Steele PJ, Pang Y, Hamilton S et al. (2012) Immunophenotype of cells within cervine rectoanal mucosa-associated lymphoid tissue and mesenteric lymph nodes. Journal of Comparative Pathology, 146, 365e371. Daszak P, Cunningham AA, Hyatt AD (2000) Emerging infectious diseases of wildlife. Threats to biodiversity and human health. Science, 287, 443e449. Drayton DL, Liao S, Mounzer RH, Ruddle NH (2006) Lymphoid organ development: from ontogeny to neogenesis. Nature Immunology, 7, 344e353. Galeotti M, Sarli G, Eleni C, Marcato PS (1993) Identification of cell types present in bovine haemolymph nodes and lymph nodes by immunostaining. Veterinary Immunology and Immunopathology, 36, 319e331. Gonzalez L, Anderson I, Deane D, Summers C, Buxton D (2001) Detection of immune system cells in paraffin wax-embedded ovine tissues. Journal of Comparative Pathology, 125, 41e47.
Mona S, Crestanello B, Bankhead-Dronnet S, Pecchioli E, Ingrosso S et al. (2008) Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois. Molecular Ecology, 17, 4053e4067. Navarro JA, Caro MR, Seva J, Rosillo MC, Gomez MA et al. (1996) Study of lymphocyte subpopulations in peripheral blood and secondary lymphoid organs in the goat using monoclonal antibodies to surface markers of bovine lymphocytes. Veterinary Immunology and Immunopathology, 51, 147e156. Pioz M, Loison A, Gauthier D, Gibert P, Julienne J et al. (2008) Diseases and reproductive success in a wild mammal: example in the alpine chamois. Oecologia, 155, 691e704. R Core Team. (2015) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/ Last access: 25/8/15. Rode B, Bavdek SV, Lackovic G, Fazarinc G, Bidovec A (1998) Immunohistochemical study of normal and mange (S. scabiei var. rupicaprae) infected chamois (Rupicapra rupicapra L.) skin. Anatomia, Histologia et Embryologia, 27, 187e192. Tingstedt J, Tornehave D, Lind P, Nielsen J (2003) Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue. Veterinary Immunology and Immunopathology, 94, 123e132. Webster JD, Miller MA, DuSold D, Ramos-Vara J (2009) Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. Journal of Histochemistry and Cytochemistry, 57, 753e761. Willard-Mack CL (2006) Normal structure, function, and histology of lymph nodes. Toxicologic Pathology, 34, 409e424.
February 11th, 2016 ½ Received, Accepted, June 28th, 2016
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra) C. Salvadori*, J. Finlayson†, T. Trogu‡, N. Formenti‡, P. Lanfranchi‡, C. Citteriox, J. Palarea-Albaladejok, A. Poli* and F. Chianini† * Department of Veterinary Sciences, Pisa University, Viale delle Piagge, Pisa, Italy, † Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Near Edinburgh, UK, ‡ Department of Veterinary Sciences and Public Health, Milan University, Via Festa del Perdono, Milano, x Istituto Zooprofilattico Sperimentale delle Venezie, Sezione di Belluno, Via M. Cappellari, Belluno, Italy and k Biomathematics and Statistics Scotland, JCMB, The King’s Buildings, Peter Guthrie Tait Road, Edinburgh, UK
Summary Immune system cell subsets in lymph nodes and spleen from alpine chamois (Rupicapra rupicapra subspecies rupicapra) living in the Italian Alps were characterized immunohistochemically. Seven primary antibodies (against human CD3, CD79acy, CD68, or ovine CD4, CD8, CD21 and gd T-cell receptor [TCR] epitopes) were tested on tissues fixed either in formalin or in zinc salts (ZS) and cross-reactivity with chamois immune cell epitopes was shown. ZS fixation allowed wider identification of immune cells, without the need for antigen retrieval. CD4+ and CD21+ cells were labelled only in ZS-fixed tissues. Reagents specific for human CD3, CD79 and CD68 antigens successfully detected chamois immune cells, both in ZS-fixed and formalin-fixed tissues. The reactivity and distribution of immune cells in lymph nodes and spleen were similar to those described in other domestic and wild ruminants. Results from this study may allow future investigation of the immune response and pathogenesis of diseases in the chamois. Ó 2016 Elsevier Ltd. All rights reserved. Keywords: alpine chamois; immune system cells; immunohistochemistry; zinc salts fixation
Alpine chamois (Rupicapra rupicapra subspecies rupicapra, Linnaeus 1758) is a wild ruminant living in the eastern Alps. Infectious diseases in chamois have raised the interest of scientists because they may represent a threat for the conservation of wildlife species and biodiversity (Daszak et al., 2000; Pioz et al., 2008). Moreover, chamois could represent a useful animal model to evaluate the complex balance between host and pathogens (especially parasites, but also viruses and bacteria). Population dynamics of chamois are influenced by pathogen-derived epidemics and investigations of the polymorphism of maCorrespondence to: F. Chianini (e-mail: Moredun.ac.uk). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2016.06.012
Francesca.Chianini@
jor histocompatibility complex (MHC) class II genes were used to study the adaptive processes and pathogen-mediated selection (Mona et al., 2008; Cavallero et al., 2012). However, evaluation of inflammatory infiltrates in tissues may be crucial to understanding of the pathogenesis of infectious diseases in this species. An immunohistochemical study of formalin-fixed tissue samples from healthy and mange-infected chamois failed to demonstrate reactivity of immune cells with a panel of antihuman antibodies (Rode et al., 1998) and no specific antibodies are available for detection of chamois immune cells. However, immunohistochemistry (IHC) is dependent on adequate fixation of the tissue. Routinely, tissues are fixed in 10% neutral buffered Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
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C. Salvadori et al.
formalin, which inhibits cellular processes and prevents tissue degradation, but is a limiting factor for IHC as it cross-links antigens and masks epitopes (Webster et al., 2009). Zinc salts (ZS) fixative is a non-aldehyde solution, composed of zinc acetate and zinc chloride in Tris-calcium acetate buffer, that is considered to preserve sensitive antigens similar to in frozen tissue, but with a morphological preservation comparable to formalin fixation (Beckstead, 1994). ZS fixation has been used successfully in immunohistochemical studies of ovine and cervine immune cells (Gonzalez et al., 2001; Dagleish et al., 2012). The aims of this study were: (1) to determine the cross-reactivity of anti-human and anti-ovine monoclonal antibodies with chamois immune cells in lymph nodes and spleen, and (2) to evaluate differences and effectiveness of immune cell labelling in chamois tissues fixed either in ZS or in formalin. Retromandibular lymph nodes and spleen were collected from 10 alpine chamois (seven females and three males), aged between 1 and 14 years, which were shot during the hunting season (from September to December 2013). A thin slice (4e5 mm thick) of each tissue sampled was immediately placed in freshly prepared ZS fixative for 24e48 h (Beckstead, 1994), and a duplicate sample was placed into 10% neutral buffered formalin for 48e72 h. All samples were processed routinely and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE). Serial sections were mounted on treated glass slides (Superfrost Plus; Menzel-Glaser, Braunschweig, Germany) for IHC. Different antigen retrieval methods were used for formalin-fixed tissues as shown in Supplementary Table 1. A panel of monoclonal antibodies (Supplementary Table 1) was applied and incubated overnight at 4 C. Antibody binding was detected by the EnVision PlusÔ System-HRP (DAB) (Dako UK Ltd., Ely, UK). Omission of the primary antibody was used to provide a negative control. Ovine lymph node sections were used as positive controls. Bright field images were acquired at 200 magnification with a Leica Microsystem DFC490 digital camera mounted on a Leica DMR microscope (Leica, Wetzlar, Germany). For each animal, five non-contiguous 10,000 mm2 fields of view were selected randomly. Labelled cells were counted in the following areas of those fields: the germinal centre and mantle zone of the follicles and the interfollicular/paracortical area and medullary cords of the lymph nodes, and the germinal centre and mantle zone of the follicles and the periarteriolar lymphoid sheaths, marginal zone and red pulp of the spleen. The number of total cells (total nuclei
evident with haematoxylin counterstaining) and CD3, CD79, CD68, CD21 and CD4-positive cells within a field were counted using a semi-automatic analysis system (LASV 4.3, Leica). The mean percentage of positively labelled cells and a 95% confidence interval computed by the AgrestieCoull method were obtained for each marker by area and fixation method. A generalized linear mixed model (GLMM) was fitted to test for significant differences in mean percentage of positive cells detected between the two fixation methods (i.e. formalin and ZS) for each organ, area and marker separately. Only markers detectable by both techniques were modelled. The GLMMs were fitted by maximum likelihood to the number of positive cells out of the total number of cells, with logit link function and binomially distributed errors, and using Laplace approximations to calculate log-likelihoods. Fixation method was included as a fixed effect and animal identification as a random effect in order to account for both within and between animal variability. The significance of the fixation method effect was assessed using P values derived from the likelihood ratio test at the usual 5% significance level. The data analyses and graphics were produced using the R system for statistical computing v3.2 (R Core Team, 2015). Good tissue morphology was evident in lymph nodes and spleen using either formalin or ZS fixation. Antibodies raised against human CD3, CD79 and CD68 epitopes reacted successfully with Alpine chamois immune cells, both in formalin- and ZSfixed tissues. Antibodies raised against ovine CD4 and CD21 labelled Alpine chamois cells only in ZSfixed tissues. Clone 7.2.38 of anti-CD3 antibody labelled the surface and cytoplasm of cells in the interfollicular/paracortical areas of lymph nodes (Supplementary Fig. 1A) and the periarteriolar lymphoid sheaths of the spleen (Supplementary Fig. 2A). A prevalence of CD3+ cells was evident in the marginal zone of the spleen and medullary cords of lymph nodes (Figs. 3 and 4). A moderate number of CD3+ cells was present in the mantle zone both in the lymph node and spleen, and few CD3+ cells were seen in the germinal centres of the follicles. No significant mean differences were detected in CD3 immunolabelling between formalin- and ZS-fixed lymph node (Fig. 1). In the spleen, significant differences were found only in the marginal zone, where ZS-fixed tissues showed a higher average percentage of labelled cells when compared with formalin-fixed tissues (64.65% versus 41.94%; P ¼ 0.024) (Fig. 2). Specific immunolabelling with anti-CD4 monoclonal antibody was observed only in ZS-fixed tissues (Supplementary Figs. 1B and 2B). CD4+ cells were
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
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Immune Cells in Fixed Tissue from Alpine Chamois Lymph nodes
CD3
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Fig. 1. Lymph nodes: mean percentages (%) of CD3 , CD79 and CD68+ cells detected by area and fixative.
lymphoid sheath of the spleen (60.97%) (Figs. 3 and 4). CD79+ cells were mainly localized in germinal centres and mantle zones of follicles in lymph nodes and spleen (Supplementary Figs. 1C and 2C).
small round cells that mirrored the distribution of CD3+ cells, although with less intense labelling. The highest mean percentages of CD4+ lymphocytes were in the interfollicular/paracortical areas of the lymph node (69.66%) and in the periarteriolar
Spleen
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Mean % positive cells
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Fig. 2. Spleen: mean percentages (%) of CD3 , CD79 and CD68 cells detected by area and fixative. PALS, periarteriolar lymphoid sheath.
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
C. Salvadori et al.
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Lymph nodes
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CD3 CD4 CD79 CD21 CD68
Mean % positive cells
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0 Germinal centre
Mantle zone
Paracortex
Medullary cords
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Fig. 3. Lymph nodes: mean percentages (%) of CD3+, CD4+, CD79+, CD21+ and CD68+ cells by area detected in ZS-fixed tissues.
Multifocally, CD79+ lymphocytes were also present in the medulla of lymph nodes and in the marginal zone of the spleen. In the medulla, labelled cells were mainly localized around small blood vessels, and multifocally, large cells with cytoplasmic label-
ling were evident. Smooth muscle cells and endothelial cells of small blood vessels occasionally reacted positively with anti-CD79 monoclonal antibody. Results of immunolabelling were variable between different areas of lymph node and spleen depending Spleen
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CD3 CD4 CD79 CD21 CD68
Mean % positive cells
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0 Germinal centre
Mantle zone
Marginal zone
PALS
Red pulp
Area
Fig. 4. Spleen: mean percentages (%) of CD3+, CD4+, CD79+, CD21+ and CD68+ cells by area detected in ZS-fixed tissues. PALS, periarteriolar lymphoid sheath.
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
Immune Cells in Fixed Tissue from Alpine Chamois
on the fixative (Figs. 1 and 2). The mean differences were significant, with formalin fixation detecting, on average, a significantly higher percentage of CD79+ cells in the germinal centres of lymph nodes (87.47% versus 68.25%; P <0.001) and spleen (91.38% versus 79.56%; P <0.001), and in the medullary cords of lymph nodes (10.92% versus 4.42%; P ¼ 0.016), and the periarteriolar lymphoid sheath (3.90% versus 1.17%; P <0.001) and the red pulp (4.36% versus 1.82%; P ¼ 0.005) of the spleen. Conversely, ZS fixation revealed the highest percentages of CD79+ cells in the interfollicular/paracortical area (4.25% versus 2.27%; P ¼ 0.003) and mantle zone (46.46% versus 38.64%; P ¼ 0.011) of the lymph node. No significant differences in the mean number of labelled cells were found for the mantle and marginal zones of the spleen. Anti-CD21 antibody labelled small round cells and, mainly, cells with a reticular pattern, in the light zone of the germinal centre and mantle zones of the lymph nodes and the splenic follicles (from 99.66% in the mantle zone of the spleen to 97.39% in the mantle zone of the lymph node) (Supplementary Figs. 1D and 2D). The distribution of CD21+ cells was similar to that of CD79+ cells; however, in all considered areas, the percentages of CD21+ cells were higher, on average, than those of CD79+ cells (Figs. 3 and 4). An intracytoplasmic granular pattern of labelling was observed for cells consistent with macrophages using monoclonal anti-human CD68 antibody (Supplementary Figs. 1F and 2F). CD68+ cells were localized mainly in the medullary cords of the lymph nodes and the marginal zone and red pulp of the spleen (Supplementary Figs. 1E and 2E). ZS fixation provided the highest average percentage of CD68+ cells in the germinal centres (12.53% versus 10.77%; P ¼ 0.040) and interfollicular/paracortical areas (7.62% versus 1.40%; P <0.001) of the lymph nodes and in the germinal centres (11.11% versus 4.38%; P <0.001) and the marginal zones (44.59% versus 35.43%; P ¼ 0.042) of the spleen. Conversely, formalin fixation revealed significantly higher mean percentages of CD68+ cells in the medullary cords (32.29% versus 20.41%; P ¼ 0.029) of the lymph nodes and the red pulp (62.45% versus 49.46%; P ¼ 0.028) of the spleen. In the other areas (i.e. the mantle zone of the lymph node and the mantle zone and periarteriolar lymphoid sheath of the spleen), there were no significant differences in the mean percentages of labelled cells between fixatives (Figs. 1 and 2). No specific labelling was detected using several clones against CD8 and gd T-cell receptor (TCR) antigens in ZS- or formalin-fixed tissues, with or without antigen retrieval. Control ovine lymph node showed
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that all antibodies labelled the expected cell populations, including CD8+ T cells and gd TCR+ cells. No labelling was detected in the negative control slides. This study provides the first description of immune system cell subsets in Alpine chamois lymph nodes and spleen. Histology of the lymph node and spleen was similar to that of other animals (Galeotti et al., 1993; Navarro et al., 1996; Gonzalez et al., 2001; Cesta, 2006; Drayton et al., 2006; Willard-Mack, 2006; Dagleish et al., 2012). The reactivity and distribution of immune cells in lymph nodes and spleen were similar to those described in goats (Navarro et al., 1996), sheep (Gonzalez et al., 2001), cattle (Galeotti et al., 1993) and deer (Dagleish et al., 2012). Monoclonal antibodies specific for human CD3, CD79 and CD68 appeared to cross-react with Alpine chamois antigens in the lymph nodes and spleen, with tissues prepared using both fixatives. Antigen-retrieval methods, which can be difficult to standardize and are time consuming, were necessary for formalin-fixed tissues in order to reverse the detrimental effects of fixation. As reported in previous immunohistochemical studies carried out on ovine and cervine tissues (Gonzalez et al., 2001; Dagleish et al., 2012), ZS fixation allowed good preservation of tissue morphology and a wider detection of cell surface epitopes compared with formalin fixation. These results could be related to the efficiency of ZS fixative, which does not cause conformational changes in the tertiary and quaternary structures of proteins, leaving the surface receptors available for the interaction with antibody. Secondary changes in the tissues after antigen retrieval treatment, which can cause rarefaction and detachment of the cells from the slides, should be taken into account in assessing the differences of immunolabelling between formalin and ZS fixation. The absence of reaction with monoclonal antibodies specific for CD8 and gd TCR was similar to results seen in cervine tissues (Dagleish et al., 2012). These antigens seem to be extremely sensitive to fixation and heating (Tingstedt et al., 2003; Dagleish et al., 2012). In conclusion, ZS appears to be an effective fixative that allows good preservation of surface receptors and specific immunolabelling for Alpine chamois lymphoid tissues. This provides a basis for future studies on the pathogenesis of diseases and immune response in this species.
Acknowledgments The authors thank M. Vascellari, Istituto Zooprofilattico Sperimentale delle Venezie, for tissue processing of ZS-fixed specimens. This work was funded by
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012
C. Salvadori et al.
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PRIN (Programmi di Ricerca di Interesse Nazionale) 2010P7LFW4_004 from the Italian Ministero dell’Istruzione, dell’Universita e della Ricerca (MIUR) and by the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS).
Conflict of Interest Statement All authors have no conflicts of interest to declare related to the publication of this manuscript.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jcpa.2016.06.012.
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February 11th, 2016 ½ Received, Accepted, June 28th, 2016
Please cite this article in press as: Salvadori C, et al., Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.012